Autosomal recessive polycystic kidney disease (ARPKD) is a significant cause of pediatric morbidity and mortality. Affected children suffer from HTN, renal insufficiency and portal tract fibrosis. The clinical spectrum of ARPKD is widely variable with most cases presenting in infancy. Our Consortium, supported by two previous awards, has used genetic approaches to define its molecular basis. In our most recent cycle, we identified the gene, PKHD1, and determined that it maps to a >400kb genomic interval, encodes a >13kb mRNA, undergoes a complicated pattern of splicing with a 67 exon transcript which encodes the longest ORF and a 4074 aa protein. We have shown that the gene is most highly expressed in kidney at all developmental stages, though it is also expressed at low levels in multiple other tissues. The gene product, polyductin, (PD), is a type I membrane protein thought likely to be a ligand or receptor. Immunolocalization studies have placed the protein in the basal body/primary cilia. In studies using epitope-tagged full length recombinant PD, we found evidence that the molecule undergoes a complicated pattern of proteolytic processing. Our genotype/phenotype studies found that individuals with biallelic truncating PKHD1 mutations have more severe disease. We isolated the mouse orthologue, determined that it also has complex splicing and generated mice with targeted mutations of the 5'end. Surprisingly, homozygous mutant mice only developed biliary and pancreatic disease. Preliminary data suggest that the targeted allele is not a true null. In this renewal, we seek to follow-up on these observations. In Aim 1, we will test the hypothesis that PD undergoes regulated proteolysis mediated by proprotein convertases, TACE or other metalloprotease, and secretase using a variety of epitope-tagged recombinant molecules, metabolic labeling, engineered mutations and mutant cell lines. We will also examine whether endogenous PD has the same properties. In Aim 2, we will develop a cell culture model system that can be used to assess the functional consequences of regulated intramembrane proteolysis. Aim 3 tests the hypothesis that complete loss of Pkhdl will result in renal and other organ dysfunction. We will characterize a newly developed line of mice with a functional floxed allele targeting exons 3 and 4. If cre-mediated deletion does not result in a complete null allele, we will generate one by introducing lox p sites flanking the entire gene. The last aim will characterize the complex pattern of splicing since this is such a prominent feature of the gene, likely explains the surprising results of recent gene targeting studies, and may account for some of the observed clinical variability.